Compositions and methods for nasal administration of drugs to brain and for systemic effect

ABSTRACT

The invention relates to a nasally administrable composition comprising at least one active substance in magnesium-containing vesicular carrier, said carrier comprising glycol, phospholipids, water and at least one magnesium source. Methods for nasal administration of the composition, for example, for pain relief, are also provided.

The present invention is directed to pharmaceutical compositions fornasal administration of biologically active agents.

Nasal delivery can provide drug absorption into the systemic circulationand it has been suggested that this route of administration can offer apathway to transport drugs the brain. There exists a need for aneffective carrier to enable enhanced nasal delivery of pharmaceuticallyactive agents.

In WO 01/06987 a carrier is disclosed, in which the major component isan ethanol/propylene glycol mixture. The carrier contains from 10 to 80%by volume of an aliphatic alcohol, 10 to 80% by volume of a glycol and0.1 to 5% by weight of a bile salt or lecithin for administeringanticonvulsive agent. The examples of WO 01/06987 illustrate a vehiclewith water content of 10%.

In U.S. Pat. No. 8,911,751, Touitou et al. describe the intranasaladministration of active ingredients to the systemic circulation orbrain with the aid of a vesicular carrier containingwater:alcohol:glycol:phospholipidsproportioned >30%:12-30%:1-30%:0.2-10%, respectively, expressed byweight percentage.

Delivery to brain of many molecules is difficult to achieve and presentsa challenge. We have surprisingly found that addition of magnesium ionto a carrier containing what we call soft phospholipid vesicles,enhances the delivery to brain and systemic action of the activemolecule.

The composition of the invention, i.e., a soft phospholipid vesicle thatcontains magnesium, displaying enhanced nasal delivery properties to thebrain and systemic action, is named herein “phospholipid magnesome”(magnesium-containing vesicular carrier).

The invention provides a nasally administrable composition comprising anactive substance (in particular a physiologically/pharmaceuticallyactive substance) in a magnesium-containing vesicular carrier, i.e., acarrier comprising glycol, phospholipids, at least one magnesium sourceand water. Hence, in its most general form, phospholipid magnesome isbased on a vesicular carrier made of glycol:phospholipids:magnesium ionand water.

The invention also provides a method of administering pharmaceuticallyactive ingredient to a mammal in need thereof, for treating variousconditions as described below, which method comprises the intranasaladministration of a composition comprising a therapeutically effectiveamount of said active ingredient in a magnesium-containing vesicularcarrier, i.e., a carrier comprising glycol, phospholipids, at least onemagnesium source and water.

The experimental results reported below indicate that themagnesium-containing vesicular carrier of the invention enhances thedelivery of drugs to the brain via the intranasal route. Accordingly,another aspect of the invention is a method of increasing the deliveryof a physiologically active compound from a nasally administrablecomposition to the brain and /or bloodstream of mammals, the methodcomprises incorporating magnesium source into a vesicular carriercomprising glycol, phospholipids, water and physiologically activecompound, e.g., CNS-active drugs.

The concentration ranges of the constituents of phospholipid magnesomeas set forth herein are by weight percentage, based on the total weightof the composition (unless indicated otherwise).

The concentration of the glycol in the composition (e.g., propyleneglycol) is not less than 5% by weight, e.g., up to 50%, for example,10-50% by weight (e.g., 10-40%). The water content of phospholipidmagnesome is not less than 20% by weight, e.g., not less than 30%, e.g.,not less than 50%.

Phospholipids suitable for use in the preparation of the compositionaccording to the present invention include phosphoglycerides, e.g.,phosphatidylcholine (lecithin, such as soy and egg lecithin). Otherphospholipids are hydrogenated phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol.Preferably, the phospholipids are present in the composition of theinvention at a concentration of 0.1 to 15% by weight, e.g., 0.2 to 10%by weight. Suitable products are phosphatidylcholine commerciallyavailable from various sources, for example, from Lipoid under the brandnames of Phospholipon®: the 85 G, 90G and 80 H, 90H grades; Lipoid®:Lipoid 100S PC, Lipoid S 100, Lipoid S 75, and others.

Suitable magnesium sources are compounds which dissociate in the liquidcarrier to release magnesium ion (Mg²⁺), e.g., pharmaceuticallyacceptable water-soluble magnesium salts such as magnesium sulfate,magnesium chloride, magnesium L-aspartate, magnesium stearate andmagnesium alginate, including their hydrated forms. The concentration ofthe magnesium salt is not less than 0.002%, e.g., from 0.005 to 5.0% byweight, preferably not less than 0.01% by weight, e.g., from 0.01 to1.0% by weight or from 0.01 to 5% by weight. But the magnesium sourcecould also be added at higher concentrations, e.g., up to 20% by weight.

Mucoadhesive agents can be added to improve adhesion of phospholipidmagnesome to the nasal mucous membrane, for example, hydroxypropylcellulose, carbomer and alginates, e.g., gel-forming agents. The desiredconcentration of the mucoadhesive agent depends on the properties of theagent selected and the desired mucoadhesivity. In general, theconcentration of this additive varies from. 0.1 to 10% by weight.

Alcohol, such as ethanol and isopropanol, is not a mandatory componentof phospholipid magnesome, but can be part of the composition, e.g., upto 25% by weight, for example, from 1 to 25% by weight. However, asshown below, the compositions are preferably free of aliphaticmonohydroxy alcohols (e.g., devoid of C2-C5 alcohols, such as ethanoland isopropanol).

Antioxidants can be added to maintain the stability of the product. Someexamples of antioxidants include tocopherols (vitamin E), butylhydroxytoluene, sodium metabisulfite, potassium metabisulfite, ascorbylpalmitate and the like. These antioxidants may be present in theformulations in a concentration of from about 0.05 to 1.0% w/w.

One method to prepare phospholipid magnesome is by dissolvingphospholipids in the glycol component, or in a mixture of glycol andalcohol, adding the physiologically active substance and the otheringredients, including of course the magnesium source, and finallycombining the glycolic/alcoholic component and the aqueous component.The magnesium source is generally added in the form of a separatelyprepared aqueous solution, but of course, it could also be employed in asolid form to be dissolved directly in the carrier.

In some cases, the total amount of water needed for the composition isdivided such that a portion, say, up to 20-30% of the total wateramount, is added in conjunction with the physiologically activesubstance or other ingredients. Some physiologically active substancesrequire basification or acidification of the medium. to facilitate theirdissolution. These physiologically active substances are firstseparately dissolved in an alkaline aqueous solution (or acidic aqueoussolution, as required) followed by respective pH adjustment, and theclear aqueous solution with the physiologically active substance ismixed with the magnesium salt solution and the remaining amount of waterand the resultant aqueous phase is combined with the phospholipidssolution to form the composition. The preparation of the composition iscarried out by mixing under various methods, homogenization or stirring,typically at room temperature or at an elevated temperature. To prepareforms of phospholipid magnesome capable of being adhered to nasal mucousmembrane, as mentioned above, a gel component (e.g., alginate, carbomer)is prepared separately, the aqueous solution of the magnesium compoundis added to the gel which is ultimately combined with the phospholipidssolution under stirring.

The experimental results reported below show that phospholipid magnesomeachieves more efficient delivery via the nasal route into mice brainthan controls. That is, a more efficient transport of an optical probe(rhodamine 6G; abbreviated R6G) via the nasal route into mice brain,that is, deeper delivery of the optical probe was measured in comparisonwith either the corresponding, magnesium-free vesicular carrier or aconventional liposome carrier. Enhanced delivery was also notedfollowing nasal delivery of insulin-labeled with fluoresceinisothiocyanate (FITC) and near-infrared (NIR) fluorescently-labeledrecombinant human epidermal growth factor (EGF).

Phospholipid magnesome has also been shown to have no negative effect ofthe nasal cavity in a rat model, e.g., mucosal epithelium remains intactwith no indication of development of inflammation following applicationof phospholipid magnesome.

Additional experimental work supporting this invention includes acomparison of the analgesic activity of a painkiller delivered eitherfrom phospholipid magnesome or from a conventional liposome (analgesicactivity of the test formulation/compounds is indicated by decrease inthe frequency of writhes in the animal model). Very good writhinginhibition was achieved with the aid of phospholipid magnesome.Pharmacokinetic study (with ketoprofen as a drug model) demonstratesrapid absorption of the drug from phospholipid magnesome via the nasalroute, as compared to significantly slower absorption from the oraladministration of an equal dose of drug.

Lastly, the transition temperature (Tm) values reported below suggeststhat phospholipid magnesome consists of soft vesicles, i.e., vesiclesexhibiting higher phospholipid fluidity as compared to correspondingconventional liposome.

In addition to the components already listed above, phospholipidmagnesome may further include auxiliary agents, such as surfactants,preservatives, thickening agents, buffers, viscosity and absorptionenhancing agents and agents capable of adjusting the pH and osmolarityof the formulation. Phospholipid magnesome may also include agents suchas tolerance enhancers to reduce or prevent drying of the mucus membraneand to prevent irritation thereof.

Suitable preservatives that can be used with phospholipid magnesomeinclude preservatives acceptable for nasal use, for example, benzylbenzalkonium salts.

Regarding buffers, phospholipid magnesome may include a buffer formaintaining the formulation at a pH of about. 7.0. The particularbuffer, of course, can vary depending upon the particular nasal deliverysystem used, as well as the specific active molecule selected. Buffersthat are suitable for use in the present invention include, for example,acetate, citrate, prolamine, carbonate and phosphate buffers andcombinations thereof. The pharmaceutical formulations of the presentinvention may include a pH adjusting agent.

Regarding thickening agents, the viscosity of phospholipid magnesome canbe maintained at a desired level using a pharmaceutically acceptablethickening agent. Thickening agents that can be added to thecompositions of the present invention include for example, methylcellulose, xanthan gum, tragacanth, adhesives, guar gum, carboxymethylcellulose, hydroxypropyl cellulose, carbomer, polyvinyl alcohol,alginates, acacia, chitosans, mucoadhesive polymer-systems likepoly(acrylates), cellulose derivatives, hyaluronic acid, hyaluronic acidderivatives, chitin, collagen, pectin, starch, poly(ethylene glycol),sulfated polysaccharides, carrageenan, Na-alginate, gelatine, pectin andcombinations thereof. The desired concentration of the thickening agentwill depend on the agent selected and the viscosity desired.

The invention also provides a nasally administrable compositioncomprising an active substance (in particular aphysiologically/pharmaceutically active substance) in a carriercomprising glycol, phospholipids, water, at least one magnesium sourceas described above and at least one oil, e.g., vegetable oil such ashemp seed oil, sesame oil or olive oil, for example, up to 5% by weight.

The compositions of the invention can be prepared as liquid, viscousliquid or gel. It can also be incorporated into different dosage formsacceptable for the nasal route of administration, e.g., they may beincorporated into various nasal creams, nasal ointments, nasalsuspensions and nasal gels in addition of course to nasal liquids.

As used herein, nasally administering or nasal administration includesadministering the compositions into naristilles of the nose to themucous membranes of the nasal passage or nasal cavity of the mammal. Forexample, the compositions of the invention can be delivered to the nasalcavity as drops; liquid delivered to the nasal cavity as non-aerosolspray (packaged in a bottle with an atomizer attachment, such as apump-sprayer) or as an aerosol spray packed in a container underpressure to emit pressurized suspension, as described in detail inRemington's Pharmaceutical Sciences (16th edition, Chapters 83 and 92)Suitable devices [nasal sprays, metered-dose sprays, squeeze bottles,liquid droppers, disposable one-dose droppers, nebulizers, cartridgesystems with unit-dose ampoules, single-dose pumps, bi-dose pumps,multiple-dose pumps] are of course commercially available from varioussources. Regarding spray devices, it should be noted that both single(unit) dose or multiple dose systems may be used. Typically, a spraydevice comprises a bottle and a pump. Typically, the volume of liquidthat is dispensed in a single spray actuation is in the range of from to250 microliters/each nostril/single administration and the concentrationof the active ingredient in the formulation may be readily adjusted suchthat one or more spray into the nostrils will comply with the dosageregimen. Administration of compositions of the present invention mayalso take place using a nasal tampon or nasal sponge containing thecompositions.

The nasal administration may be used for systemic delivery of activecompounds through the circulation or for CNS delivery for treatingCNS-originating diseases or conditions.

A wide range of physiologic active substances can be administered viathe nasal route with the aid of phospholipid magnesome to treat one ormore of the following diseases and conditions: neurological disorder,muscular disturbances, ticks, brain, CNS, insomnia, pain, anxiety,migraine, glioma, epilepsy, astoglioma, cancer, acne, IBD, Chron'sdisease, loss of appetite, fear, distress, panic, tremor, multiplesclerosis, autism, Alzheimer, menopause, Parkinson, awakens, good mood,post-traumatic, alcoholic and nonalcoholic fatty liver, hysteria,seizure and types of encephalopathy, including hepatic-encephalopathy.

Phospholipid magnesome is, under a preferred aspect of the invention,suitable for enhancing the transport of hydrophilic compounds of anymolecular size, inter alia, peptides, proteins and hydrophilic smallmolecule compounds (in the form of pharmaceutically acceptable salts),and nucleic acid based molecules such as antisense, iRNA, siRNA, microRNA and anti-microRNA to the brain or systemically, as indicated by theresults reported below. That is, peptides, amino acids, proteins andsteroid hormones (e.g. insulin, insulin derivatives, insulin detemir,insulin monomeric, oxytocin, LHRH, LHRH analogues, adreno-corticotropichormone, somatropin, leuprolide, calcitonin, parathyroid hormone,estrogens, testosterone, adrenal corticosteroids, megestrol,progesterone, sex hormones, growth hormones, growth factors, etc.)

But other active compounds can benefit from being delivered fromphospholipid magnesome, for example cannabinoids and in particularderivatives of cannabinoids that have been rendered hydrophilic. Thecannabinoid compound, either natural or synthetic, may be utilized in asolid form or in the form of an extraction concentrate, solvent extract,oil extract and oil solution, possibly surfactant-containing extractsand solutions. A. non-limiting list of cannabinoids is given below:

Δ⁹-THC, available under the name dronabinol; and Δ⁸-THC.

CBD (chemical named2-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenedi-ol).The synthesis of CBD was described, for example, by Gaoni Y, Mechoulam R[Tetrahedron Letters. 26 (8): 1083-1086 (1985)]; and by Petilka et al.[Helv. Chim. Acta, 52:1102 (1969); and in J. Am. Chem. Soc., 87:3273(1965)].

CBN (chemically named6,6,9-trimethyl-3-pentyl-6H-dibenzo[b,d]pyran-1-ol). The synthesis ofCBN was described by Novak et al., Tetrahedron Letters, 23:253 (19(32);and by Jesse A. Teske and Alexander Deiters Org. Lett., 2008, 10 (11),pp 2195-2198.

Nabilone (chemically named:3-(1,1-dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-1-hydroxy-6,6-dimethyl-9-H-dibenzo[b,d]pyran-9-one).The preparation of this synthetic cannabinoid is described, for example,in U.S. Pat. No. 3,968,125.

Levonantradol (chemically named: (−)-(6S,6aR,9R,10aR)-5,6,6a,7,8, 9,10,10a-octahydro-6-methyl-3-[(R)-1-meth-yl-4-phenylbutox]-1,9--phenanthridinediol1-acetate. The preparation of this synthetic cannabinoid is described,for example, in U.S. Pat. Nos. 4,206,225, 4,232,018, 4,260,764,4,235,913, 4,243,674, 4,263,438, 4,270,005, and 4,283,569.

(−)-HU-210 (chemically named:(−)-(3S,4S)-7-hydroxy-Δ⁶-tetrahydrocannabinol-1,1-dimethylhept-yl). Thepreparation of this synthetic cannabinoid can is found in U.S. Pat. Nos.4,876,276 and 5,521,215.

(+)-HU-210 (chemically named:(+)-(3S,4S)-7-hydroxy-Δ⁶-tetrahydrocannabinol-1,1-dimethylhept-yl). Thepreparation of this synthetic cannabinoid is described in U.S. Pat. Nos.4,876,276 and 5,521,215.

11-hydroxy-Δ⁹-THC, which can be prepared via the synthetic routedescribed by Siegel et al., J. Org. Chem., 54:5428 (1989).

Δ⁸-tetrahydrocannabinol-11-oic acid, which is naturally occurringderivative and can be produced synthetically employing methods describedin U.S. Pat. No. 6,162,829.

CP 55,940 (chemically named: 4-(1,1-dimethylheptyl)-2,3′dihydroxy-6′alpha-(3-hydroxypropyl)-1′,2′,3′,4′,5′,6′-hexahydrobiphenyl),which is commercially available from Tocris Cookson, Inc., Itspreparation has been described; see for example U.S. Pat. Nos. 4,371,720and 4,663,474.

R(+)-WIN 55,212-2 (chemically named:(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1-,4-benzoxazin-6-yl]-1-naphthalenyl-methanone)is commercially available in the form of its mesylate salt from variousmanufacturers.

Crude herbal cannabis—in countries and jurisdictions where it is, orwill become, legally allowed—can also be delivered using the compositionof this invention.

It should be noted that the compositions of the invention is not limitedto the delivery of a single active ingredient, and it may be used toprovide combination therapy, that is, a second active ingredient couldbe added to the composition.

The following active ingredients (and of course, pharmaceuticallyacceptable salts thereof) can also be incorporated into phospholipidmagnesome:

-   Antimalarial agents (e.g. artemisinin derivatives,    dihydroartemisinin, artemotil, chloroquine, primaquine, doxycillin,    quinine, aminoquinolines, cinchona alkaloids, antifolates,    quinidine, melfoquine, halofantrine, lumefantrine, amodiaquine,    pyronaridine, tafenoquine, artesunates, artemether, artemotil,    biguanides, proguanil, chloproguanil, diaminopyrimidines,    pyremethamine, trimethoprim, dapsone, sulfonamides, atovaquone,    sulfadoxine-pyrimethamine, N-acetyl cysteine, piperaquine,    DHA-piperaquine, lumefantrine, dermaseptins, bisphosphonates,    quercitin etc. The drugs could be used alone or in combinations.-   OTC drugs (e.g. antipyretics, anesthetics, cough suppressants, etc.)-   Antiinfective agents-   Antibiotics (e.g. penicillins, cephalosporins, macrolides,    tetracyclines, aminoglycosides, anti-tuberculosis agents,    doxycycline, ciprofloxacine, moxifloxacine, gatifloxacine,    carbapenems, azithromycine, clarithromycine, erythromycine,    ketolides, penems, tobramyicin, filgrastim, pentamidine, microcidin,    clerocidin; amikacine, etc.)-   Genetic molecules (e.g. Anti-sense oligonucleotides, nucleic acids,    oligonucleotides, DNA, RNA, iRNA, siRNA, micro RNA and    anti-microRNA)-   Anti-cancer agents (e.g. anti-proliferative agents,    anti-vascularization agents, taxol, etopside, cisplatin, etc.)-   Anti-protozoal agents-   Antivirals (e.g. acyclovir, gancyclovir, ribavirin, anti-HIV agents,    anti-hepatitis agents, famciclovir, valaciclovir, didanosine,    saquinavir, ritonavir, lamivudine, stavudine, zidovudine, etc.)-   Anti-inflammatory drugs (e.g. NSAIDs, steroidal agents,    cannabinoids, leukotriene-antagonists, tacrolimus, sirolimus,    everolimus, etc.)-   Anti-allergic molecules (e.g. antihistamines, fexofenadine)-   Bronchodilators-   Vaccines and other immunogenic molecules (e.g. tetanus toxoid,    reduced diphtheria toxoid, acellular pertussis vaccine, mums    vaccine, smallpox vaccine, anti-HIV vaccines, hepatitis vaccines,    pneumonia vaccines, influenza vaccines, TNF-alpha-antibodies etc.)-   Anesthetics, local anesthetics.-   Antipyretics (e.g. paracetamol, ibuprofen, diclofenac, aspirin,    etc.)-   Agents for treatment of severe events such cardiovascular attacks,    seizures, hypoglycemia, etc.-   Afrodisiacs from plants or synthetics-   Anti-nausea and anti-vomiting.-   Immunomodulators (immunoglobulins, etc.)-   Cardiovascular drugs (e.g. beta-blockers, alpha-blockers, calcium    channel blockers, etc.)-   Peptide and steroid hormones (eg. insulin, insulin derivatives,    insulin detemir, insulin monomeric, oxytocin, LHRH, LHRH analogues,    adreno-corticotropic hormone, somatropin, leuprolide, calcitonin,    parathyroid hormone, estrogens, testosterone, adrenal    corticosteroids, megestrol, progesterone, sex hormones, growth    hormones, growth factors, etc.)-   Peptide and protein related drugs (e.g. amino acids, peptides,    polypeptides, proteins)-   Vitamins (e.g. Vit A, Vitamins from B group, folic acid, Vit. C, Vit    D, Vit E, Vit K, niacin, derivatives of Vit D, etc.)-   Autonomic Nervous System Drugs-   Fertilizing agents-   Antidepressants (e.g. buspirone, venlafaxine, benzodiazepins,    selective serotonin reuptake inhibitors (SSRIs), sertraline,    citalopram, tricyclic antidepressants, paroxetine, trazodone,    lithium, bupropion, sertraline, fluoxetine, etc.)-   Agents for smoking cessation (e.g. bupropion, nicotine, etc.)-   Agents for treating alcoholism and alcohol withdrawal-   Lipid-lowering agents (e.g. inhibitors of 3    hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase,    simvastatin, atrovastatin, etc.)-   Drugs for CNS or spinal cord (benzodiazepines, lorazepam,    hydromorphone, midazolam, Acetaminophen, 4′-hydroxyacetanilide,    barbiturates, anesthetics, etc.)-   Anti-epilepsic agents (e.g. valproic acid and its derivatives,    carbamazepin, etc.)-   Angiotensin antagonists (e.g. valsartan, etc.)-   Anti-psychotic agents and anti-schizophrenic agents quetiapine,    risperidone)-   Agents for treatment of Parkinsonian syndrome (e.g. L-dopa and its    derivatives, trihexyphenidyl, etc.)-   Anti-Alzheimer drugs (e.g. cholinesterase inhibitors, galantamine,    rivastigmine, donepezil, tacrine, memantine, N-methyl D-aspartate    (NMDA) antagonists).-   Agents for treatment of non-insulin dependent diabetes metformine,-   Agents against erectile dysfunction (e.g. sildenafil, tadalafil,    papaverine, vardenafil, PGE1, etc.)-   Prostaglandins-   Agents for bladder dysfunction (e.g. oxybutynin, propantheline    bromide, trospium, solifenacin succinate etc.)-   Agents for treatment menopausal syndrome (e.g estrogens,    non-estrogen compounds, etc.)-   Agents for treatment hot flashes in postmenopausal women-   Agents for treatment primary or secondary hypogonadism (e.g.    testosterone, etc.)-   Cytokines (e.g. TNF, interferons, IFN-alpha, IFN-beta, interleukins    etc.)-   CNS stimulants-   Muscle relaxants-   Anti paralytic gas agents-   Appetite stimulators/depressors (e.g. cannabinoids, etc.)-   Gastrointesinal absorption modifiers-   Narcotics and Antagonists (e.g. opiates, oxycodone etc.)-   Painkillers (opiates, endorphins, tramadol HCl, codein, NSAIDs,    gabapentine, fentanil and pharamaceutically acceptable salts thereof    etc.)-   Hypnotics (Zolpidem, benzodiazepins, barbiturates, ramelteon, etc.)-   Histamines and Antihistamines-   Antimigraine Drugs (e.g. imipramine, propranolol, sumatriptan, eg.)-   Diagnostic agents (e.g. Phenolsulfonphthalein, Dye T-1824, Vital    Dyes, Potassium Ferrocyanide, Secretin, Pentagastrin, Cerulein,    etc.)-   anti-inflammatory drugs-   ADHD related medication (e.g. methylphenidate, dexmethylphenidate,    dextroamphetamine, d- and l-amphetamin racemic mixture, pemoline,    etc.)-   Diuretic agents-   Anti-osteoporotic agents (e.g. bisphosphonates, aledronate,    pamidronate, tirphostins, etc.)-   Drugs for treatment of asthma-   drugs for post trauma, crisis, anxiety treatment-   Anti-Spasmotic agents (e.g. papaverine, etc.)-   Agents for treatment of multiple sclerosis and other    neurodegenerative disorders (eg. mitoxantrone, glatiramer acetate,    interferon beta-1a, interferon beta-1b, etc.)-   Plant derived agents from leave, root, flower, seed, stem or    branches extracts.-   Anti anxiety drugs.

The experimental work reported below shows that the carrier of theinvention can be used for delivery to the brain via the nasal route ofpharmaceutically active agents spanning a wide range of molecular weightand physiochemical properties, e.g.:

-   -   large molecules (e.g., molecular weight >1000 g/mol) such as        peptide hormones (insulin, oxytocin) and proteins.    -   small molecules, for example, with molecular weights up 500        g/mol including compounds bearing hydrophilic functional groups        (carboxylic acid, hydroxyl) and salt-forming groups, e.g.,        compounds administered as acid addition salts, such as tramadol        hydrochloride (analgesic), butorphanol tartrate (opioid);        rizatriptan benzoate (antimigraine) safinamide mesylate        (anti-Parkinson); and other small molecules such as mannitol,        ketoprofen and brotizolam, and also cannabinoids (CBD, CBN, THC        or mixtures thereof).

Accordingly, specific aspects of the invention include compositionscomprising a pharmaceutically active agent selected from:

an analgesic, antimigraine and/or antipyretic agent (e.g., tramadol,ketoprofen, rizatriptan and pharmaceutically acceptable salts thereof);

an opioid (e.g., butorphanol and pharmaceutically acceptable saltsthereof);

an anti-Parkinson drug (e.g., safinamide and pharmaceutically acceptablesalts thereof);

a sedative and/or hypnotic drug (e.g., brotizolam or a pharmaceuticallyacceptable salt thereof);

a peptide or a polypeptide peptide hormone selected from the groupconsisting of insulin and oxytocin, or bacitracin);

a cannabinoid or a mixture of cannabinoids (CBD, THC, CBN or a mixturethereof).

It should be noted that concentration of the active agent in thecomposition of the invention may vary broadly, from 0.01 to 20% byweight.

Additional aspect of the invention relates to a method of administeringan active substance to a mammal in need thereof, comprising intranasaladministration of a composition comprising a therapeutically effectiveamount of the active substance (e.g., an analgesic, antimigraine and/orantipyretic agents; an opioid; an anti-Parkinson drug; a sedative and/orhypnotic drug; peptide or a polypeptide; a cannabinoid) in amagnesium-containing vesicular carrier that contains glycol,phospholipids, water and at least one magnesium source.

Use of a vesicular carrier that contains glycol, phospholipids, waterand at least one magnesium source as described herein (e.g., monohydroxyaliphatic alcohol-free carrier), for intranasal administration ofpharmaceutically active agents for systemic delivery or CNS (centralnervous system) delivery, is another aspect of the invention.

Owing to its ability to offer a rapid onset of an analgesic effect, thecomposition of the invention is especially useful in a method for painrelief, the method comprising the intranasal administration to a patientof a composition comprising a carrier and therapeutically effectiveamount of an active substance selected from the group consisting of ananalgesic, an opioid, an antimigraine agent and an anti-inflammatory,wherein the carrier in a magnesium-containing vesicular carriercomprising glycol, phospholipids, water and at least one magnesiumsource.

We have also found that the pharmaceutically active agent pramipexolecould be administered intranasally with the aid of themagnesium-containing vesicular carrier of the invention to achieveefficient therapeutic effect. Pramipexole a dopamine agonist drug withselective activity at D2/D3-receptors. It is approved as mono andadjunctive therapy for treatment of motor symptoms in patients withParkinson's disease. Currently pramipexole was administrated orally inimmediate and extended release tablet formulations. Transdermal deliveryof pramipexole was described in U.S. Pat. No. 5,112,842. Little has beenreported on the nasal delivery of pramipexole, e.g., pramipexole wasloaded on chitosan nanoparticles [raj et. al., In J Biol Macromol 20181; 109:27-35].

The commercial form of pramipexole is its dihydrochloride salt (of the(S)-enantiomer). The term pramipexole, as used herein, is directed to2-amino-6-propylamino-4,5,6,7-tetrahydrobenzothiazole, its(S)-enantiomer, and pharmaceutically acceptable salts thereof, such asthe dihydrochloride salt. However, it also includes the (R)-enantiomerand mixtures of both. Utilities of the (R)-enantiomer are reported inU.S. Pat. No. 7,157,480.

Studies reported herein indicate greatly improved results of behavioraltesting in Parkinson's mice model following nasal administration ofpramipexole by the composition of the invention. That is, significantreversal of reserpine-induced locomotor impairment, reserpine-inducedptosis and reserpine-induced catalepsy compared to pramipexole oralsolution. Pramipexole used in the studies reported below was pramipexoledihydrochloride monohydrate.

Another aspect of the invention is therefore a method of treatingParkinson's disease, motor symptoms associated with Parkinson's disease,including treating impaired locomotion in Parkinson's disease patients,Parkinsonism, and other pramipexole-treatable diseases, such as restlesslegs syndrome and other diseases and conditions affected by dopaminemodulation), which method comprises administering intranasally, to amammal in need thereof, a pharmaceutical composition comprisingpramipexole and a vesicular carrier.

The invention further provides a method for treating impairedlocomotion, comprising administering intranasally, to a patient in needthereof, a pharmaceutical composition comprising anti-Parkinson drug(such as pramipexole), in a vesicular carrier.

The term vesicular carrier, as used herein in connection with theadministration of pramipexole, is a carrier comprising phospholipids,water and one of monohydroxy/dihydroxy alcohol, or both. However, thepreferred vesicular carrier is the magnesium-containing vesicularcarrier disclosed herein, namely, Phospholipid Magnesome, whichcomprises glycol, phospholipids, water and at least one magnesium source(and is generally free of monohydroxy alcohol).

Accordingly, another aspect of the invention is a nasally administrablecomposition comprising pramipexole in magnesium-containing vesicularcarrier, said carrier comprising glycol, phospholipids, water and atleast one magnesium source, e.g., from 0.1 to 2% by weight pramipexole(e.g., from. 0.5 to 1.0%), from 5 to 50% by weight propylene glycol(e.g., from 10 to 30%), from 0.2 to 10% by weight phospholipids (e.g.,from 1 to 5%), not less than 20% by weight water (e.g., not less than50%) and not less than 0.01% magnesium source (e.g., from 0.05 to 1.0%by weight magnesium salt such as MgSO₄). The composition may furthercontain an antioxidant such as vitamin E and an alkaline agent such assodium hydroxide.

The therapeutically effective amount of pramipexole is generally from0.1 to 5 mg (e.g., from 0.001 to 0.05 mg/kg of body weight).Experimental work reported below indicates that nasally-administrablecompositions containing from 0.5 to 1.0% by weight of pramipexole arereadily formulated, such that administration of one or more applications(e.g., spray, drops, gel) into the nostrils 1 to 6 times per day willcomply with the dosage regimen.

Lastly, it should he noted that the nasal vesicular composition withoutan added drug/pharmaceutically active compound, can also be used, e.g.,for ONS effect. The carrier of the invention may contain highconcentration of magnesium, useful for such purpose. Hence nasallyadministrable vesicular composition comprising glycol, phospholipids,water and at least one magnesium source, wherein the composition isdevoid of an active substance, constitutes another aspect of theinvention. Concentration ranges for the components are as previouslyindicated.

In the drawings:

FIG. 1: Three-dimensional micrographs of the olfactory region for micebrain treated with R6G at a dose of 10 mg/kg from phospholipidsmagnesome, water solution and Liposome. Height: 589 μm, width: 589 μm,depth: 606 μm, lens ×20 (A1-MP microscope NIKON-Japan).

FIG. 2: a bar diagram showing fluorescent intensity (A.U.) in theolfactory region of mice brain at 10 min after treatment with R6G at adose of 3 mg/kg in phospholipids magnesome, water solution and Liposome;Mean±SD. Auto fluorescence was subtracted. **P value<0.01 consideredvery significant.

FIG. 3: Three-dimensional micrographs of the olfactory region for micebrain treated with R6G at a dose of 10 mg/kg from phospholipid magnesomevs. soft vesicles. Height: 979 μm, width: 979 μm, depth: 507 μm, lens×20 (A1-MP microscope NIKON-Japan).

FIG. 4: a bar diagram showing fluorescent intensity (A.U.) in theolfactory region of mice brain at 10 min after treatment withInsulin-FITC at a dose of 1 mg/kg in phospholipids magnesome, WS andLipo; Mean±SD. Auto fluorescence was subtracted. **P value<0.01considered very significant.

FIG. 5: NIR images for mice brains treated with EPG IRDye 800CW at adose 1mg/Kg from phospholipid magnesome as compared to water solutionand liposome.

FIG. 6: a bar diagram showing fluorescent intensity (A.U.) in theolfactory region of mice brain at 10 min after treatment with EGF-IRDye800CW at a dose of 1 mg/kg in phospholipids magnesome, as compared towater solution and liposome; Mean±SD. Auto fluorescence was subtracted.**P value<0.01 considered very significant.

FIG. 7: a bar diagram showing the writhing counts for mice treated withOxytocin at a dose of 0.4 mg/kg phospholipid magnesome, WS, Lipo andUntreated control 5, 30 and 120 min prior to IP injection of aceticacid; Mean±SD. P<0.05 for phospholipid magnesome vs. untreated, WS andLipo 5 min. P<0.01 for phospholipid magnesome vs. untreated, WS and Lipoat 30 and 120 min time points.

FIGS. 8A-8D: Representative micrographs of nasal cavities excised fromrats that (A) received no treatment or treated with (B) phospholipidMagnesome, (C) NS and (D) SLS nasal solution.

FIGS. 9A-9B: DSC thermograms for (A) phospholipids magnesome, (B)liposome obtained by Mettler Toledo DSC-1 STAR system (China).

FIG. 10: a graph showing the pharmacokinetic profile of Ketoprofen inplasma following nasal administration of Ketoprofen from the compositionof the invention vs. Ketoprofen oral administration, each at a dose of14 mg/Kg. Results (mean±SD) **P<0.01, very significant at 10 min, and 30min.

FIG. 11: Number of squares crossed in the open field test by Parkinson'smice treated with:

Nasal Pramipexole Phospholipid Magnesome (n=6);

Oral Pramipexole solution (n=4) and

untreated animals (n=5) , (Mean ±SD). p<0.001 for PramipexolePhospholipid Magnesome vs. untreated control, p<0.01 for PramipexolePhospholipid Magnesome vs. oral, p>0.05 (considered not significant) forPramipexole oral vs. untreated control by one-way ANOVA.

FIG. 12: Ptosis scores for Parkinson's mice model treated with:

Nasal Pramipexole Phospholipid Magnesome (n=6);

Oral Pramipexole Solution (n=4) and

untreated animals (n=5), (Mean±SD). p<0.01 for Pramipexole PhospholipidMagnesome vs. untreated control, p>0.05 for Pramipexole nasal vs. oraland oral vs. untreated, by one-way ANOVA.

FIG. 13: Number of squares crossed in the open field test by Parkinson'smice model treated with:

Nasal Pramipexole Phospholipid Magnesome;

Oral Pramipexole solution and

untreated animals (n=7/group), (Mean ±SD). p<0.001 for PramipexolePhospholipid Magnesome vs. untreated control, p<0.01 for PramipexolePhospholipid Magnesome vs. oral, p>0.05 (considered not significant) forPramipexole oral vs. untreated control by one-way ANOVA.

FIG. 14. Catalepsy duration measured by bar test for Parkinson's micemodel treated with:

Nasal Pramipexole Phospholipid Magnesome;

Oral Pramipexole solution and

untreated animals (n=7/group), (Mean ±SD). p<0.001 for PramipexolePhospholipid Magnesome vs. untreated control, p<0.01 for PramipexolePhospholipid Magnesome vs. oral, p>0.05 (considered not significant) forPramipexole oral vs. untreated control by two tail Mann-Whitney test.

FIG. 15. Ptosis scores for Parkinson's mice model treated with:

Nasal Pramipexole Phospholipid Magnesome;

Oral Pramipexole solution and

untreated animals (n=7/group), (Mean ±SD). p<0.001 for PramipexolePhospholipid Magnesome vs. untreated control, p<0.01 for nasal vs. oraland p>0.05 for oral vs. untreated, by one-way ANOVA.

EXAMPLES

Abbreviations used in the examples:

-   PL—Phospholipid-   PG—Propylene Glycol-   WS—Water solution-   DDW—Double distilled water-   ETOH—Ethanol-   HSO—Hemp Seed Oil-   SO—Sesame oil-   Lipo—Liposome

The following materials were used: Magnesium Sulfate, anhydrous fromJ.T. Baker, USA; Phospholipon 90 G—Lipoid, Phospholipid GmbH, Germany;Propylene Glycol—Tamar, Israel and Vitamin E Acetate—Tamar, Israel.

Example 1 Nasal Delivery to Brain

Nasal delivery of R6G in the composition of the invention to theolfactory region of mice brain visualized by a Multiphoton Microscope(A1-MP microscope NIKON-Japan).

Part I

Nine female C57B1/6J mice (8-9 weeks), were divided equally into 3groups of administration. The following formulations were prepared:

Phospholipid magnesome Water solution Liposome (% w/w) % w/w % w/w R6G 11 1 PL 3 — 3 PG 15 — — Sodium alginate 0.6 — 0.6 Magnesium sulfate 0.03— — DDW To 100 To 100 To 100

Preparation: PL was dissolved in PG, R6G was added and then 20% of thetotal DDW amount was added, through mixing. In a separate vessel, SodiumAlginate was dispersed in 10% of the total amount of DDW and mixed withMagnesium Sulfate aqueous solution (10 mg/ml). The PL solution was thenadded to the alginate gel through mixing with an overhead stirrer(Heidolph digital 200 RZR-2000, Germany), then the remaining DDW wasadded.

R6G was administered nasally to mice from the three above formulationsat a dose of 10 mg/kg animal (˜0.2 mg\20 μl/mouse). Ten minutes aftertreatments, the animals were sacrificed, the brains were removed, washedwith normal saline and examined by the Multiphoton Microscope using thefollowing conditions: excitation λ 850 nm, field of image of 589×589×606nm (width×height×depth), lens ×60 laser, intensity 5%, scanning 512,scan speed 0.5, line skipping 2, Luts1500 and zoom1. The fluorescenceintensity of the probe (arbitrary units A.U.) in the olfactory region inbrain was further assessed using Image Pro-Plus software.

The micrographs are presented in FIG. 1 (left: phospholipid magnesome;middle: water solution; right: liposome). FIG. 2 is a bar diagramshowing the fluorescent intensity. The micrographs of MultiphotonMicroscopic examination and the semi-quantification showed that higherfluorescent signals are found in the olfactory region in the groupstreated with phospholipid magnesome containing R6G relative to differentcontrol nasal compositions Water Solution (WS) and Liposome (Lipo). Itis worthy to notice that the fluorescence intensity in the WS group wasapparently higher (yet not statistically significant) than that achievedin the animals receiving Lipo treatment.

These results point towards the efficiency of phospholipid magnesome toimprove the hydrophilic probe delivery to the examined region in thebrain.

Part II

In this part, the effect of pospholipid magnesome was evaluated incomparison with a composition without magnesium.

Female C57B1/6J mice (8-9 weeks), were divided into 2 groups ofadministration. The following formulations were prepared:

Phospholipid magnesome No Mg (% w/w) % w/w R6G 1 1 PL 3 3 PG 15 15Magnesium sulfate 0.03 — DDW To 100 To 100

The administered dose and the experimental procedure was performed asdescribed in Part I of this example.

FIG. 3 shows the three-dimensional micrographs. The results of this partof the experiment show deeper delivery of R6G into the examined brainregion. The semi quantification indicated a fluorescence of 13.2 A.U. tothe phospholipid magnesome as compared to 8.0 A.U. for the softvesicles, such results pointing towards the superiority of phospholipidmagnesome as carrier for brain delivery of drugs.

Example 2 Nasal Delivery of Insulin-FITC to Brain

Nasal delivery of Insulin FITC to the olfactory region in mice brainfrom the composition of the invention and two control compositions,examined by a Multiphoton Microscope (A1-MP microscope NIKON-Japan).

Nine female C57B1/6J mice (8-9 we were divided equally into groups ofadministration. The following formulations were prepared.

Phospholipid magnesome Water solution Liposome (% w/w) % w/w % w/wInsulin FITC 0.1 0.1 0.1 PL 3 — 3 PG 15 — — Sodium alginate 0.6 — 0.6Magnesium sulfate 0.03 — — DDW To 100 To 100 To 100

Preparation: PL was dissolved in PG, Insulin FITC was added then 20% ofthe total DDW amount was added, through mixing. In a separate vessel,Sodium Alginate was dispersed in 10% of the total amount of DDW andmixed with Magnesium Sulfate aqueous solution (10 mg/ml). The PLsolution was then added to the alginate gel through mixing and then theremaining DDW was added.

Insulin FITC was administered nasally to mice from the threeformulations at a dose of 1 mg/kg animal (˜0.02 mg\20 μl/mouse). Tenminutes after treatments, the animals were sacrificed, the brains wereremoved, washed with normal saline and examined by the MultiphotonMicroscope using the following conditions: excitation λ of 860 nm, field58.6×58.6×30.6 nm (width×height×depth), lens ×20, laser intensity 11.1%,scanning 512, scan speed 0.5, no line skipping, Luts: 1000 and zoom 9.9.The fluorescence intensity of the probe (arbitrary units A.U.) in theolfactory region in brain was further assessed using Image Pro-Plussoftware.

Multiphoton imaging of the olfactory region following Insulin-FITCadministration in phospholipid magnesome indicates the presence ofaugmented fluorescent signal in this group relative to controls(micrographs are not shown). Semi-quantification of the fluorescencesignals gave fluorescent intensities of ˜24 A.U. in brain section ofanimals treated with phospholipid magnesome containing Insulin-FITC ascompared to 3.4 and 7.0 A.U. for the controls WS and Lipo, respectively,as shown in the bar diagram of FIG. 4.

Example 3 Nasal Delivery of EGF-IRDye 800CW to Brain

Nasal delivery of EGF-IRDye 800CW to brain of mice from phospholipidmagnesome as compared with two controls, was examined by Odyssey®Infrared Imaging System (LI-OCR, USA).

Twelve female C57B1/6J mice (8-9 weeks) were divided equally into 3groups of administration and Untreated Control group. The followingformulations were prepared.

Phospholipid magnesome Water solution Liposome (% w/w) % w/w % w/wEGF-IRDye 800CW 0.1 0.1 0.1 PL 3 — 3 PG 15 — — Sodium alginate 0.6 — 0.6Magnesium sulfate 0.03 — — DDW To 100 To 10 0 To 100

Preparation: PL was dissolved in PG, EGF-IRDye 800CW was added and then20% of the total DDW amount was added, through mixing. In a separatevessel, Sodium Alginate was dispersed in 10% of the total amount of DDWand mixed with Magnesium Sulfate aqueous solution (10 mg/ml). The PLsolution was then added to the alginate gel through mixing, then theremaining DDW was added.

EGF-IRDye 800CW was administered nasally to mice from the threeformulations at a dose of 1 mg/kg animal (˜0.02 mg\20 μl/mouse). Tenminutes after treatments, the animals were sacrificed; brains wereremoved, washed with normal saline and observed under the imagingsystem. The scanning was performed using offset 3, resolution 339.6 μm,channel 800 nm and intensity 3.

The NIR images (FIG. 5; from left to right: untreated group,phospholipid magnesome-treated group; water solution-treated group andliposome-treated group) and their semi-quantification (FIG. 6, in theform of a bar diagram) indicate the presence of the labeled peptidesignal in brain tissues following nasal administration from varioussystems as compared to Untreated Control. FIG. 5 shows that followingadministration from phospholipid magnesome, EGF-IRDye 800CW accumulatedin the cerebrum and in the olfactory bulb with remarkable accumulationin the cerebrum. The administration from water solution lead to peptideaccumulation in the olfactory bulb (FIG. 5). The fluorescent signalscalculated to be 19.1 A.U. in phospholipid magnesome containingEGF-IRDye 800CW as compared to 10.0 and 6.7 A.U. in WS and Lipo,respectively (FIG. 6).

Example 4 Analgesic Effect of Oxytocin Nasally Delivered in PhospholipidMagnesome as Compared to Two Control Carriers

The analgesic effect of intranasal administration of Oxytocin inphospholipid magnesome composition was evaluated in female C57B1/6Jmice. The acetic acid-induced pain mice model was used.

In this model, animals received analgesic treatment, then afterpredetermined time periods, pain was induced by 0.6% (v/v) acetic acidsolution injected intraperitoneally at a dose of 10 ml/kg. The number ofwrithes in a 10 min period was counted, starting 5 min after the aceticacid injection. A writhe is characterized by a wave of contraction ofthe abdominal musculature followed by extension of at least one hindlimb. Antinociception is expressed as percent inhibition of the numberof writhes observed in treated animals in comparison to animals in theuntreated group.

The Maximum Possible Effect (MPE) of different treatments is expressedas the inhibition percent of the number of writhes in a drug-treatedanimal group, when compared to the mean number of writhes measured in agroup of untreated control mice according to the following equation.

MPE %=[Mean of writhes in untreated control group−Mean of writhes intreated group]/[Mean of writhes in untreated control group]*100

Twenty mice were divided into three equal treatment groups for testingthree time points: 5, 30 or 120 min (n=5/group) and Untreated controlgroup. The following formulations were prepared.

Phospholipid magnesome Water solution Liposome (% w/w) % w/w % w/woxytocin 0.08 0.08 0.08 PL 3 — 3 PG 15 — — Sodium alginate 0.6 — 0.6Magnesium sulfate 0.03 — — DDW To 100 To 100 To 10 0

Preparation: PL was dissolved in PG, Oxytocin was added and then 20% ofthe total DDW amount was added, through mixing. In a separate vessel,Sodium Alginate was dispersed in 10% of the total amount of DDW andmixed with Magnesium Sulfate aqueous solution (10 mg/ml). The PLsolution was then added to the alginate gel through mixing, then theremaining DDW was added.

Oxytocin was administered nasally to mice from the three formulations ata dose of 0.4 mg/kg animal (˜0.008 mg\10 μl/mouse).

FIG. 7 and the table below give the writhes counts and MPE % values,respectively, following treatment of mice with 0.4 mg/kg Oxytocinincorporated in various carriers. As shown in the bar diagram of FIG. 7,a significant decrease in writhes number was observed followingtreatment with Oxytocin from phospholipid magnesome (the darkest bar).The effect of the peptide incorporated in various controls (WS and Lipo)was lower by 2 folds, underscoring the enhanced delivery of Oxytocinfrom phospholipid magnesome.

Below are tabulated calculated MPE % values following Oxytocinadministration to mice at a dose of 0.4 mg/kg from phospholipidmagnesome and control nasal systems administrated at various time pointsbefore pain induction.

Phospholipid magnesome Water solution Liposome Time (min) MPE % 5 63.535.8 30.8 30 63.9 27.8 25.8 120 58.0 29.8 19.2

Example 5 Local Safety

The effect of phospholipid magnesome on the nasal cavity was evaluatedin rats. Female SD; H. rats were divided into four groups:

-   Group 1—(phospholipid magnesome): Intranasal administration of    phospholipid magensome composition as described in Example 1, Part    II (15 μl/rat)-   Group 2—(NS): Intranasal Normal saline (15 μl/rat).-   Group 3—(SLS): Intranasal Sodium lauryl sulfate Solution (1% w/w),    (15 μl/rat).-   Group 4—Untreated control animals.

The animals received the treatments twice a day for one week. At the endof the experiment, animals were sacrificed, nasal cavities were removedand fixed in 3.7% Formaldehyde PBS. Sections of the nasal cavity werecut serially at 7 μm thickness and stained with Hematoxylin & Eosin. Thesections were examined by professional histopathologist (Authority forAnimal Facilities, Hebrew University of Jerusalem, Israel) by Olympuslight microscope BX43 and Olympus digital camera DP21 with OlympuscellSens Entry 1.13 software (Olympus, Japan) using magnification ×10.Local toxicity was assessed by evaluating the histopathologicalalterations in different regions of the nasal cavity (cartilage andturbinate bone, lamina propria and submucosa, mucosal epithelium andlumen).

No pathological findings were observed in the histopathological analysisof the nasal cavities excised from rats treated with phospholipidmagnesome or NS. The micrographs for these groups were similar tountreated control group showing intact mucosal epithelium, empty lumenand no infiltration of inflammatory cells. Overall, there was noevidence of inflammation. Turbinate bone integrity was preserved.Epithelium was normal with no evidence of erosion or ulceration andciliated epithelium was intact. On the other hand, minimal proteinaceousmaterial in the lumen and focal aggregations of neutrophils wereobserved in the positive control group treated with SLS. Micrographscorresponding to the four groups are presented in FIG. 8.

Example 6 Tramadol HCl-Containing Phospholipid Magnesome

Ingredients % w/w Tramadol HCl 10 PL 3 PG 15 Sodium Alginate 0.6Magnesium Sulfate 0.01 DDW To 100

Preparation: PL was dissolved in PG, Tramadol and 20% of the total DDWamount were added, through mixing. In a separate vessel, Sodium Alginatewas dispersed in 10% of the total amount of DDW and mixed with MagnesiumSulfate aqueous solution (10 mg/ml). The PL solution was then added tothe alginate gel through mixing with an overhead stirrer (Heidolphdigital 200 RZR-2000, Germany), then the remaining DDW was added.

Example 7 Rizatriptan Benzoate-Containing Phospholipid Magnesome

Ingredients % w/w Rizatriptan Benzoate 10 PL 3 PG 15 Magnesium Sulfate0.03 DDW To 100

Preparation: PL was dissolved in PG, then Rizatriptan was added, throughmixing. To this mixture, Magnesium Sulfate aqueous solution (10 mg/ml)was added through mixing with a magnetic stirrer, then the remaining DDWwas added.

Example 8 Mannitol-Containing Phospholipid Magnesome

Ingredients % w/w Mannitol 10 PL 3 PG 15 Magnesium Sulfate 0.07 DDW To100

Preparation: PL was dissolved in PG, then Mannitol was dispersed in thePL solution. To this mixture Magnesium Sulfate aqueous solution (10mg/ml) was added through mixing with an overhead stirrer (Heidolphdigital 200 RZR-2000, Germany), then DDW was added.

Example 9 Cannabidiol-Containing Phospholipid Magnesome

Ingredients % w/w CBD 1 PL 3 PG 25 Ethanol Absolute (ETOH) 15 MagnesiumSulfate 0.01 DDW To 100

Preparation: PL was dissolved in ETCH and PG mixture, then CBD wasdissolved in the PL solution. To this solution, Magnesium Sulfateaqueous solution (10 mg/ml) was added through mixing with a magneticstirrer then DDW was added.

Example 10 Cannabidiol-Containing Phospholipid Magnesome

Ingredients % w/w CBD 0.5 PL 5 PG 25 ETOH 17 Magnesium Sulfate 0.01 DDWTo 100

The composition is prepared with a high shear mixer. PL was dissolved inETOH and PG mixture, then CBD was dissolved in the PL solution. To thissolution, Magnesium Sulfate aqueous solution (10 mg/ml) was addedthrough mixing with a magnetic stirrer then DDW was added.

Example 11 Brotizolam-Containing Phospholipid Magnesome

Ingredients % w/w Brotizolam 0.1 PL 3 PG 15 Carbopol 980 0.05 Ammoniumhydroxide 0.05 Magnesium Sulfate 0.02 DDW To 100

PL was dissolved in PG. Brotizolam was added to the solution. In aseparate vessel, Carbopol 980 was suspended in DDW and ammoniumhydroxide was added. Then Magnesium Sulfate aqueous solution (10 mg/ml)was added to this mixture followed by adding the PL solution throughmixing with an overhead stirrer (Heidolph digital 200 RZR-2000,Germany).

Example 12 Cannabidiol-Containing Phospholipid Magnesome

Ingredients % w/w CBD 1 PL 10 PG 40 Vit E 0.4 Magnesium Sulfate 0.03 DDWTo 100

PL was dissolved in PG, then CBD and Vit E were dissolved in the PLsolution. To this solution, Magnesium Sulfate aqueous solution (10mg/ml) was added through mixing with a magnetic stirrer then DDW wasadded.

Example 13 Butorphanol Tartrate (BUT)-Containing Phospholipid Magnesome

Ingredients % w/w BUT 0.1 PL 2 PG 30 Sodium Hydroxide 0.5 MagnesiumSulfate 0.025 DDW To 100

Preparation: PL was dissolved in PG, then BUT was dissolved in the PLsolution. To this solution, DDW and magnesium sulfate solutions wereadded through mixing with an overhead stirrer (Heidolph digital 200RZR-2000, Germany). Finally, Sodium Hydroxide was added (for pHadjustment). Final pH—5.5.

Example 14 Ketoprofen (KET)-Containing Phospholipid Magnesome

Ingredients % w/w KET 20 PL 2 Magnesium Sulfate 0.025 PG 30 SodiumHydroxide (10%) 40 Hydrochloric Acid (32%) 5.8 DDW To 100

Preparation: PL was dissolved in PG. In a separate vessel, KET wassuspended in 20% of DDW, then Sodium Hydroxide Solution was added (todissolve KET) and followed by the addition of Hydrochloric Acid (for pHadjustment) and the rest amount of DDW and magnesium sulfate solutionwee added. Finally, the PL solution was added to this solution and mixedwith an overhead stirrer (Heidolph digital 200 RZR-2000, Germany).

Example 15 Ketoprofen (KET)-Containing Phospholipid Magnesome

Ingredients % w/w KET 3 PL 2 PG 30 Sodium Hydroxide (10%) 10 Magnesiumsulfate 0.02 HCl solution (32%) 2 DDW To 100

Preparation: PL was dissolved in PG. In a separate vessel, KET wassuspended in 20% of DDW, then Sodium Hydroxide Solution was added (todissolve KET) and followed by the addition of Hydrochloric Acid (for pHadjustment). The Magnesium solution, and the rest amount of DDW wereadded. Finally, the PL solution was added to this solution and mixedwith an overhead stirrer (Heidolph digital 200 RZR-2000, Germany). FinalpH—5.7

Example 16 Assessment of Phospholipid Magnesome Softness (More FluidBilayers Relative to Liposome) by Transition Temperature (Tm)Measurement by Differential Scanning Calorimetry (DSC)

Tm of the phospholipid was measured in the following compositions:

phospholipid magnesome Liposome Ingredients % w/w % w/w Magnesiumsulfate 0.03 — PL 5 5 PG 15 — DDW To 100 To 100

The measurements were carried out using a Mettler Toledo DSC-1 STARsystem (Toledo, China). Samples of 20 mg were placed in aluminum metaldishes. Tomograms were generated recording Tm values at a heating rateof 10° C./min within the temperature range of −50° C. to +50° C.

Results indicate that phospholipid magnesome systems had a Tm value of−7° C. vs. +7° C. for liposome. This lower Tm by 14° C. could be theresult of a fluidization of the PL lamellae in phospholipid magnesomevesicles n comparison with classic liposome. The thermograms are shownin FIGS. 9A and 9B.

Example 17 Assessment of Drug Concentration in Plasma andPharmacokinetic Parameters

Plasma concentration of Ketoprofen was measured following in vivo nasaladministration and compared to oral administration. The experiment wascarried out using Male Sprague Dawley (St /Hsd) rats (Harlan, Israel).

The method of Ketoprofen extraction from plasma was validated accordingto FDA regulations for bio-analytical method validation, assessingprecision, recovery, selectivity and linearity. The precision was 8.7%,the recovery was 96.6±5.5%, the limit of detection (LOD) was 0.84mcg/ml, and the limit of quantification (LOQ) was 2.53 mcg/ml, Linearregression analysis of the plasma standard curve showed correlation withR²=1.00 over the concentration range of 0.05-100 mcg/ml Ketoprofen. Thecalibration curve equation: Y=143453X, (Y=Area, X=Concentration).

Ketoprofen (14 mg/kg) was administered nasally from Ketoprofen newnanovesicular carrier (KET-phospholipid Magnesome) and compared to oraladministration (KET-PO). The compositions are tabulated below.

Compositions KET -phospholipid magnesome KET-PO Ingredients % w/w % w/wKetoprofen (KET) 20 2 PL 5 — PG 30 — Sodium Hydroxide (10%) 40 40Hydrochloric Acid (32%) 5.8 5.8 Magnesium sulfate 0.02 — MethylCellulose — 2 DDW To 100 To 100

Preparation: PL is dissolved in PG. In a separate vessel, KET issuspended in a part of water, then Sodium Hydroxide Solution is added(to dissolve KET) and followed by the addition of hydrochloric acid (forpH adjustment). The magnesium solution and the rest amount of DDW areadded. Finally, the PL solution is added to this aqueous solution andmixed with an overhead stirrer (Heidolph digital 200 RZR-2000, Germany)or Polytron homogenizer. Final pH—5.4

Blood samples were collected from rats' tails at 10, 30, 60, 120, 180,240 and 300 min post drug administration. The blood samples werecentrifuged at 3k rpm for 10 min at 25° C. (HERMLE Z 160 M), and then150 mcl of the plasma was taken. Plasma samples were frozen and kept at−20° C. until analysis.

A volume of 300 mcl of ACN was added to the plasma and mixed by vortexfor 3 minutes at level 10, followed by the addition of 300 mcl ofacetate buffer 0.05M with. pH 5, and mixed by vortex for additional 1minute at level 10. Then, the samples were centrifuged for 5 min at 14krpm. at 25° C. (HERMLE Z 160 M), and the supernatants were filteredthrough Bulk GHP Acrodisc® 13 mm syringe filter with 0.45 um GHPmembrane (Pall Corporation, USA), and transferred into pre-labeled autoinjector vials before being injected into HPLC-UV.

The relative bioavailability (F %) was calculated according to thefollowing equation:

The AUC_(NVC) and AUC_(PO) represent the means of individual AUC fromnasal and oral experimental groups, respectively.

The pharmacokinetic study aimed to evaluate the influence of the newnanovesicular carrier on the absorption parameters of the drug model,Ketoprofen. For this purpose, Ketoprofen concentration was assessed inplasma of rats following drug administration from new nanovesicularcarrier as compared to oral administration. Ketoprofen plasmaconcentration curves versus time are plotted in FIG. 10 (rectangular:nasal administration according to the invention; triangle oraladministration.

Ketoprofen was assayed in rat plasma starting from 10 min post nasal ororal administration. Results are tabulated below.

PK parameters Nasal composition Oral administration T_(1/2), min 138.2 ±21.1 328.7 ± 83.1  Tmax, min 10.0 ± 0.0 94.0 ± 12.4 Cmax, mcg/ml 43.7 ±8.3 13.7 ± 5.9  AUC_(0-5 hr) 4130.4 ± 730.6 2642.1 ± 1153.3 T_(last),min 300.00 ± 0.00   300 ± 0.00 Bioavailability 156.3 (relative to oral,%)

Results presented above show that very significant higher plasmaconcentrations (P<0.01) were detected 10 and 30 min after Ketoprofenadministration in nasal vesicular nanocarrier. C_(max) plasma valuescalculated for new nanovesicular carrier and oral administration were43.65±8.30 and 13.68±5.91 mcg/ml, respectively. The T_(max) values were10 and 94 min for nasal vesicular carrier administration and oraladministration, respectively.

These results above indicate that Ketoprofen was rapidly delivered fromthe nasal cavity to the systemic circulation following nasaladministration showing that the phospholipid magnesome possessesenhanced delivery properties and enhanced effect for the first 90minutes relative to the oral administration, with a very rapid onset ofaction and behaves similar to the oral administration for the next fivetested hours.

Example 18 Cannabidiol-Containing Phospholipid Magnesome

Ingredients % w/w CBD 5 PL 5 PG 30 Ethanol 15 Hemp Seed Oil (HSO) 4Magnesium Sulfate 0.03 DDW To 100

PL is dissolved in PG and Ethanol mixture, and then HSO is added. CBD isdissolved in this solution. Then, Magnesium Sulfate aqueous solution (10mg/ml) is slowly added through vigorous mixing with a homogenizer.Finally, DDW is slowly added with mixing.

Example 19 Cannabidiol-Containing Phospholipid Magnesome

Ingredients % w/w THC 0.5 PL 5 Vit E 0.5 PG 30 Olive Oil 3 MagnesiumSulfate 0.03 DDW To 100

PL is dissolved in PG, then Vit E, Oil and THC are added to the PLsolution. To this solution, Magnesium Sulfate aqueous solution (10mg/ml) is added slowly through mixing with an overhead stirrer (Heidolphdigital 200 RSR-2000, Germany).

Examples 20-21 Insulin-Containing Phospholipid Magnesome

Example 20 Example 21 ingredients ingredients % w/w % w/w Insulin 0.10.1 PL 3.0 3.0 PG 15.0 15.0 Vitamin E 0.3 0.3 Magnesium sulfate 1.0 5.0DDW 80.6 76.6

Preparation: PL was dissolved in PG, then Vitamin E was added. In aseparate vessel, Magnesium Sulfate was dissolved in DDW. The Magnesiumsolution was added gradually to the PL solution and mixed well. Finally,Insulin (from Bovine Pancreas-Sigma Aldrich, USA) was added and mixedwell. The mixing through the entire preparation process was performedusing an overhead Heidolph® stirrer (Heidolph Digital 200 RZR-2000,Germany),

Examples 22-23 Bacitracin-Containing Phospholipid Magnesome

Example 22 Example 23 ingredients ingredients % w/w % w/w Bacitracin 0.10.1 PL 3.0 3.0 PG 15.0 15.0 Vitamin E 0.3 0.3 Magnesium sulfate 10 20DDW 71.6 61.6

PL was dissolved in PG, then Vitamin E was added. In a separate vessel,Magnesium Sulfate was dissolved in DDW. The Magnesium solution was addedgradually to the PL solution and mixed well. Finally, Bacitracin (SigmaAldrich, USA) was added and mixed well. The mixing through the entirepreparation process was performed using an overhead Heidolph® stirrer(Heidolph Digital 200 RZR-2000, Germany).

Example 24 Tramadol HCl-Containing Phospholipid Magnesome

ingredients % w/w Tramadol HCl 1.0 PL 3.0 PG 15.0 Vitamin E 0.3Magnesium sulfate 5.0 DDW 75.7

PL was dissolved in PG, then Vitamin E was added. In a separate vessel,Tramadol HCl (Chemagis, Israel) was dissolved in DDW and followed bydissolving the Magnesium Sulfate. The aqueous solution of Tramadol HCland Magnesium was added gradually to the PL solution and mixed well. Themixing through the entire preparation process was performed using anoverhead Heidolph® stirrer (Heldoiph Digital 200 RZR-2000, Germany).

Example 25 Safinamide Mesylate-Containing Phospholipid Magnesome

ingredients % w/w Safinamide mesylate 2.0 PL 5.0 PG 15.0 Vitamin E 0.5Magnesium sulfate 0.5 DDW to 100

PL is dissolved in PG, then Vitamin E is added. In a separate vessel,Safinamide mesylate is dissolved in DDW and is added gradually to the PLsolution and mixed well using an overhead Heidolph® stirrer (HeidolphDigital 200 RZR-2000, Germany). Finally, the Magnesium Sulfate is addedto the composition and mixed.

Examples 26-34 Phospholipid Magnesome with Varying Mg Content

Ex. 26 Ex. 27 Ex. 28 Ex. 29 Ex. 30 Ex. 31 Ex, 32 Ex. 33 Ex. 34ingredients % w/w %w/w % w/w % w/w % w/w % w/w % w/w % w/w % w/w PL 3.03.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 PG 15.0 15.0 15.0 15.0 15.0 15.0 15.015.0 15.0 Vitamin E 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.3 0.3 Magnesiumsulfate 0.01 0.1 0.3 0.5 1.0 2.0 5.0 10.0 20.0 DDW 81.69 81.6 81.4 81.280.7 79.7 76.7 71.7 61.7

PL was dissolved in PG, then Vitamin E was added. In a separate vessel,Magnesium Sulfate was dissolved in DDW. The Magnesium solution was addedgradually to the PL solution and mixed well using an overhead Heidolph®stirrer (Heidolph Digital 200 RZR-2000, Germany).

Example 35 Pramipexole-Containing Phospholipid Magnesome

ingredients % w/w Pramipexole 1.00 Propylene glycol (PG) 20.0Phospholipon 90 G (PL) 3.00 α-D- tocopheryl acetate (Vit E) 0.50Magnesium sulfate anhydrous (MgSO₄) 0.10 Sodium hydroxide (NaOH) 0.05Double distilled water (DDW) To 10 0

PL was mixed with PG using an overhead stirrer at 700 rpm (Heidolph, HeiTorque 200) until completely dissolved. Vit E was added and mixed well.MgSO₄ was dissolved in about one third of the water amount, and thesolution was added to the above PG solution through mixing.

In a separate vessel, NaOH 1% w/v solution was added to two thirds ofthe water amount. Pramipexole was dissolved in the above water solution.pH was measured and, if needed, adjusted to ˜4.5 with NaOH. ThisPramipexole solution was then added through mixing at 700 rpm to theabove system. The mixing was further continued for 5 min.

Example 36 Pramipexole-Containing Phospholipid Magnesome

ingredients % w/w Pramipexole 0.50 Propylene glycol (PG) 20.0Phospholipon 90 G (PL) 3.00 α-D-tocopheryl acetate (Vit E) 0.50Magnesium sulfate anhydrous (MgSO₄) 0.10 Sodium hydroxide (NaOH) 0.023Double distilled water (DDW) To 100

PL was mixed with PG using an overhead stirrer at 700 rpm (Heidolph, HeiTorque 200) until completely dissolved. Vit E was added and mixed well.MgSO₄ was dissolved in about one third of the water amount, and thesolution was added to the above PG solution through mixing.

In a separate vessel, NaOH 1% w/v solution was added to two thirds ofthe water amount. Pramipexole was dissolved in the above water solution.pH was measured and, if needed, adjusted to ˜4.5 with NaOH. ThisPramipexole solution was then added through mixing at 700 rpm to theabove system. The mixing was further continued for 5 min.

Example 37 Effect of Nasal Administration of Pramipexole PhospholipidMagnesome Versus Drug Oral Administration in Mice Model for ParkinsonDisease with Locomotor Impairment

The goal of the experiment reported below was to evaluate the effect ofnasal administration of Pramipexole Phospholipid Magnesome on impairedlocomotor activity in model mice for Parkinson's disease in comparisonwith oral administration of the drug and untreated animals. The animalmodel was obtained by administering Reserpine to mice.

Experimental Protocol

Compositions

The compositions tested were the one illustrated in Example 36 (0.5% w/wPramipexole in Phospholipid Magnesome) and an aqueous solution of 0.5%w/w Pramipexole in water for oral administration, prepared by addingNaOH 1% w/v solution to DDW to achieve NaOH at concentration of 0.022%w/w, followed by dissolution of the drag in the alkaline solution.

Animals

All procedures carried out on animals were according to The NationalInstitutes of Health regulations and were approved by the Committee forAnimal Care and Experimental Use of the Hebrew University of Jerusalem.

The experiment was performed on fifteen male CD-1 ICR mice (27-32 g).Mice were housed under standard conditions of light and temperature inplastic cages in the specific-pathogen unit (SPF) of the pharmacy schoolat the Hebrew University of Jerusalem. Animals were kept in separatedcages with smooth flat floor and provided with unlimited access to waterand food.

Treatments

The mice were divided randomly into two drug treated groups, PramipexolePhospholipid Magnesome administrated nasally (n=6), Pramipexole oralsolution (n=4) and one untreated control group (n=5). Animals in thetreatment groups received Pramipexole nasally from PhospholipidMagnesome or orally from solution at a dose of 3 mg/kg. Twenty minutesafter the treatments, the behavioral testing was assessed. To rule outthe effect of anesthesia, animals in the untreated control groups wereanesthetized at the same time points before the behavioral testing.

On the first and eighth days of the experiment, the animals in the threegroups received intraperitoneal injections of Reserpine at doses of 4and 3 mg/kg, respectively. Reserpine injection was prepared in DDWcontaining 0.1% DMSO and 0.3% Tween 80. The suspension was furtherprocessed for 20 min at 50% power ratio using an Ultrasonic processor,Sonic-650WT-V2 Ultrasonic processor. Sonic Series, MRC Ltd, Holon,Israel.

Behavioral Testing

The behavioral tests (open field test and ptosis score) were performedon day 9 of the experiment, 23 hours after last Reserpine injection and20 min following nasal or oral Pramipexole administration.

Open Field Test

Spontaneous locomotor activity of animals was measured using the openfield test. Mice were placed in the center of a cage (29×28.5×30 cm),with the floor divided into nine equal squares. The number of squarescrossed was counted during 5 min with no habituation session.

Normal animal moves in the cage and crosses squares on the floor.Reserpinized animal suffers from akinesia (cannot move) and crosses muchless squares. Efficient treatment will reverse animal's behavior tonormal.

Ptosis Score

Ptosis is the eye closure due to drooping of the upper eyelid. Reserpineinduced ptosis was visually determined. Ptosis was recorded on a 0-4scale, in which 0 represents eyes completely shut, and 4 completelyopen.

Normal animal has a ptosis score of 4. The score for reserpinizedanimals is reduced to 0-1. Efficient treatment will reverse the score tonormal.

Results of the Open Field Test

The number of squares crossed (Mean ±SD) in the open field test byParkinson's mice model treated are tabulated below.

Nasal Pramipexole Phospholipid Pramipexole Group Magnesome OralUntreated Number of 81.8 ± 25.3 27.5 ± 30.1 2.6 ± 3.6 squares crossed*Normal animals cross more than 100 squares.

The results are also presented graphically in the form of a bar diagramin FIG. 11.

The results pertaining to the open field test indicate that mice treatednasally with Pramipexole Phospholipid Magnesome expressed higherlocomotor activity and crossed 81.8±25.3 squares. The animals in thePramipexole orally treated and the untreated groups crossed only27.5±30.1 and 2.6±3.6 squares, respectively (FIG. 11). The results showthat the nasal administration of Pramipexole Phospholipid Magnesomesignificantly (p<0.01) enhanced the locomotor activity by 300% incomparison with oral treatment.

Results of Ptosis Score

Ptosis scores for Parkinson's mice model treated (Mean±SD) are tabulatedbelow.

Nasal Pramipexole Phospholipid Pramipexole Group Magnesome oralUntreated Ptosis score 3.3 ± 0.7 2.0 ± 1.4 1.2 ± 0.4 *Normal animal hasa ptosis score of 4.

The results are also presented graphically in the form of a bar diagramin FIG. 12.

In the evaluation of Reserpine-induced ptosis, a score of 3.3±0.7 wasrecorded for nasal. Pramipexole Phospholipid Magnesome as compared toonly 2.0±1.4 and 1.2±0.4 for the orally treated and the untreatedgroups, respectively (FIG. 12).

These results indicate enhanced anti-Parkinson's effect of Pramipexoleachieved by means of nasal administration using Phospholipid Magnesomein comparison with oral administration.

Example 38 Effect of Nasal Administration of Pramipexole PhospholipidMagnesome Versus Drug Oral Administration in Mice Model for ParkinsonDisease with Locomotor Impairment

The goal of the experiment was to evaluate the effect of nasaladministration of Pramipexole Phospholipid Magnesome on impairedlocomotor activity in model mice for Parkinson's disease in comparisonwith oral administration of the drug and untreated animals. The animalmodel was obtained by administrating Reserpine to mice.

Experimental Protocol Compositions

The compositions tested were the one illustrated in Example 36 (0.5% w/wPramipexole in Phospholipid Magnesome) and an aqueous solution of 0.5%w/w Pramipexole in water for oral administration, prepared by addingNaOH 1% w/v solution to DDW to achieve NaOH at concentration of 0.022%w/w, followed by dissolution of the drag in the alkaline solution.

Animals

All procedures carried out on animals were according to The NationalInstitutes of Health regulations and were approved by the Committee forAnimal Care and Experimental Use of the Hebrew University of Jerusalem.

This experiment was performed on 21 male CD-1 ICR mice (25-29 g). Micewere housed under standard conditions of light and temperature inplastic cages in the specific-pathogen unit (SPF) of the pharmacy schoolat the Hebrew University of Jerusalem. Animals were kept in separatedcages with smooth flat floor and provided with unlimited access to waterand food.

Treatments

The mice were divided randomly into two drug treated groups (PramipexolePhospholipid Magnesome administrated nasally, and pramipexole oralsolution) and one untreated control group (n=7/group). Animals in thetreatment groups received Pramipexole at a dose of 3 mg/kg nasally fromPhospholipid Magnesome or Pramipexole orally from solution, 20 minbefore the behavioral testing. To rule out the effect of anesthesia,animals in the untreated control groups were anesthetized at the sametime points before the behavioral testing.

On the first and sixth days of the experiment, the animals of the threegroups received an intraperitoneal injections of Reserpine at a dose of3 mg/kg.

Behavioral Testing

The behavioral tests (open filed test, ptosis score and bar catalepsytest) were performed on day 7 of the experiment, 23 hours after lastReserpine injection. Open field test and ptosis score were assessed 20min following nasal or oral Pramipexole administration. Bar catalepsytest was carried out immediately after the open field test (25 minfollowing Pramipexole administration).

Open Field Test

The same protocol as in Example 37.

Ptosis Score

The same protocol as in Example 37.

Bar Catalepsy Test

Cataleptic immobility is regarded as an animal equivalent of akinesiaand is demonstrated by an animal allowing its body to be placed in andmaintain abnormal or unusual postures. We used the bar test to determinethe ability of nasal administration of Pramipexole in PhospholipidMagnesome to reduce the duration of catalepsy in Parkinson's mice modelin comparison with oral administration of the drug.

Twenty-five minutes after nasal and oral treatments, both fore paws ofthe mice were placed on a horizontal bar (diameter, 0.7 cm) 5 cm abovethe surface and then gently released. The catalepsy duration retained inthis unusual position was recorded in seconds from the moment when ananimal was released to the moment when it shifted its front paws fromthe initial position on the bar. The trial ended either when the animalstarted to move or after 60 s of immobility (cut off time).

Normal animal releases the bar immediately. Reserpinized animal suffersfrom catalepsy cannot return to normal position and therefore stays onthe bar for longer time. Efficient treatment will reverse animal'sbehavior to normal.

Results of the Open Field Test

The number of squares crossed (Mean±SD) in the open field test byParkinson's mice model treated are tabulated below.

Nasal Pramipexole Phospholipid Pramipexole Group Magnesome oralUntreated Number of 109.0 ± 29.8 37.4 ± 22.5 28.5 ± 32.4 squarescrossed. *Normal animals cross more than 100 squares.

The results are also presented graphically in the form of a bar diagramin FIG. 13.

The results pertaining to the open field test indicate that mice treatednasally with Pramipexole Phospholipid Magnesome expressed higherlocomotor activity and crossed 109.0±29.8 squares. The animals in thePramipexole orally treated and the untreated groups crossed only37.4±22.5 and 28.5±32.4 squares, respectively (FIG. 13). The results aresignificant: p<0.01 for Pramipexole Phospholipid Magnesome versusPramipexole oral and p<0.001 for Pramipexole Phospholipid Magnesomeversus untreated control.

Results of the Bar Catalepsy Test

Catalepsy duration by the bar test (Mean±SD) for Parkinson's mice modeltreated are tabulated below.

Nasal Pramipexole Phospholipid Pramipexole Group Magnesome oralUntreated Catalepsy 0.8 ± 0.6 9.0 ± 8.3 21.3 ± 18.7 duration (sec)*catalepsy duration measured by bar test for normal animals is 0 sec.

The results are also presented graphically in the form of bar diagram inFIG. 14.

The results pertaining to the bar catalepsy test indicate that nasaladministration of Pramipexole from Phospholipid Magnesome reduced thecatalepsy duration after 25 min from 21.3±18.7 in the reserpinizeduntreated animals to 0.8±0.6 sec (p<0.001). Oral administration of thesame dose led to mild and non-significant reduction in the catalepsyduration (9.0±8.3 sec).

Results of Ptosis Score

Ptosis scores for Parkinson's mice model treated (Mean±SD) are tabulatedbelow.

Nasal Pramipexole Phospholipid Pramipexole Group Magnesome oralUntreated Ptosis score 3.7 ± 0.5 1.7 ± 1.1 1.4 ± 1.0 *Normal animal hasa ptosis score of 4.

The results are also presented graphically in the form of a bar diagramin FIG. 15.

In the evaluation of Reserpine induced ptosis, a score of 3.7±0.5 wasrecorded for Pramipexole Phospholipid Magnesome as compared to only1.7±1.1 and 1.4±1.0 for the orally treated and the untreated groups,respectively (FIG. 15). The results of these experiments confirm thefinding illustrated in Example 37 and show that the nasal administrationof Pramipexole Phospholipid Magnesome significantly increased thelocomotor activity by 300% in comparison with oral treatment.Furthermore, the nasal treatment completely reversed the reserpineinduced catalepsy. These finding point towards the ability of nasaladministration of Pramipexole Phospholipid magnesome to improve thetreatment of Reserpine induced Parkinson's in mice.

1) A nasally administrable composition comprising at least one activesubstance in magnesium-containing vesicular carrier, said carriercomprising glycol, phospholipids, water and at least one magnesiumsource. 2) The composition of claim 1, comprising from 5 to 50% byweight propylene glycol, from 0.2 to 10% by weight phospholipids, notless than 20% by weight water and not less than 0.01% by weightmagnesium source. 3) The composition of claim 2, comprising from 0.01 to20.0% by weight magnesium salt. 4) The composition of claim 3,comprising from 0.01 to 5.0% weight magnesium salt. 5) The compositionof claim 1, further comprising an antioxidant, a mucoadhesive agent orboth. 6) The composition according to claim 1, which is free ofaliphatic monohydroxy alcohol. 7) The composition according to claim 5,wherein the magnesium source is magnesium sulfate and the mucoadhesiveagent is an alginate salt. 8) The composition according to claim 1,further comprising a vegetable oil. 9) The composition of claim 1,wherein the active substance is an analgesic, antimigraine and/orantipyretic agent. 10) The composition of claim 9, wherein theanalgesic, antimigraine and antipyretic agent is selected from the groupconsisting of tramadol, ketoprofen, rizatriptan and pharmaceuticallyacceptable salts thereof. 11) The composition of claim 1, wherein theactive substance is an opioid. 12) The composition of claim 11, whereinthe opioid is butorphanol or a pharmaceutically acceptable salt thereof.13) The composition of claim 1, wherein the active substance is ananti-Parkinson drug. 14) The composition of claim 13, wherein theanti-Parkinson drug is selected from. the group consisting ofsafinamide, pramipexole and pharmaceutically acceptable salts thereof.15) The composition of claim 1, wherein the active substance is asedative and/or hypnotic drug. 16) The composition of claim 15, whereinthe sedative and/or hypnotic drug is brotizolam or a pharmaceuticallyacceptable salt thereof. 17) The composition of claim 1, wherein theactive substance is a peptide or a polypeptide. 18) The compositionaccording to claim 15 comprising peptide hormone selected from the groupconsisting of insulin and oxytocin, or bacitracin. 19) The compositionaccording to claim 1, wherein the active substance is mannitol. 20) Amethod of administering an active substance to a mammal in need thereof,comprising intranasal administration of a composition comprising atherapeutically effective amount of the active substance in amagnesium-containing vesicular carrier that contains glycol,phospholipids, water and at least one magnesium source. 21) A methodaccording to claim 20, wherein the composition further comprises anantioxidant, a mucoadhesive agent or both. 22) A method according toclaim 20, wherein the composition is free of aliphatic monohydroxyalcohol. 23) A method according to claim 20, wherein the activesubstance is selected from the group consisting of an analgesic,antimigraine and/or antipyretic agents; opioids; an anti-Parkinson drug;a sedative and/or hypnotic drug. 24) A method according to claim 20,wherein the active substance is selected from the group consisting ofpeptide or polypeptides. 25) A method of increasing the delivery of aphysiologically active compound from a nasally administrable compositionto the bloodstream/brain of mammals, the method comprises incorporatingmagnesium source into a vesicular carrier which contains glycol,phospholipids, water and said physiologically active compound, forenhanced delivery. 26) A method according to claim 25, wherein theactive compound is selected from the group consisting of an analgesic,antimigraine and/or antipyretic agents; an opioid; an anti-Parkinsondrug; a sedative and/or hypnotic drug; and a hormone peptide. 27) Amethod for pain relief, comprising the intranasal administration to apatient of a composition comprising a carrier and therapeuticallyeffective amount of an active substance selected from the groupconsisting of an analgesic, an opioid, an antimigraine agent, andanti-inflammatory agent, wherein the carrier in a magnesium-containingvesicular carrier comprising glycol, phospholipids, water and at leastone magnesium source. 28) A method of treating Parkinson's disease,symptoms associated with Parkinson's disease or Parkinsonism, whichmethod comprises administering intranasally, to a patient in needthereof, a pharmaceutical composition comprising pramipexole and avesicular carrier. 29) A method according to claim 28, for treatingimpaired locomotion in Parkinson's disease patients. 30) A methodaccording to claim 28, wherein the carrier is a magnesium-containingvesicular carrier comprising glycol, phospholipids, water and at leastone magnesium source. 31) A method for treating impaired locomotion,comprising administering intranasally, to a patient in need thereof, apharmaceutical composition comprising anti-Parkinson drug in a vesicularcarrier. 32) A method for treating Restless Leg Syndrome, comprisingadministering intranasally, to a patient in need thereof, apharmaceutical composition comprising pramipexole in a vesicularcarrier. 33) A nasally administrable vesicular composition comprisingglycol, phospholipids, water and at least one magnesium source, whereinthe composition is devoid of an active substance.